Infection with enterohemorrhagic E. coli (EHEC), acquired from contaminated food or water, represents a major health problem worldwide with EHEC being the fourth most costly foodborne pathogen in the USA. Although many EHEC strains produce Shiga toxin (Stx), its role in pathogenesis is not understood. In some species, the presence of attaching and effacing (A/E) lesions correlate more closely with intestinal symptoms than does the expression of Stx. While the formation of A/E lesions may be sufficient to induce diarrhea, the production of Stx may be a prerequisite for hemorrhagic colitis. These findings underscore the lack of understanding of the pathogenesis of EHEC. While much effort has been focused on the extraintestinal manifestations of EHEC, the effects of this important pathogen on the intestine have been grossly understudied. The hypothesis of this proposal is that EHEC has direct effects on its initial host target tissue, the intestinal epithelium, which contribute to the associated symptoms. The studies proposed here will utilize two separate models to investigate the effects of EHEC infection on intestinal epithelial function, a reductionist model of cultured human intestinal epithelia and a murine model. Preliminary data show that two major physiological processes, tight junction (TJ) barrier function and the epithelial-initiated inflammatory cascade, are altered in both of these models following infection with EHEC. It is likely that both of these alterations contribute to EHEC-associated diarrhea. The impact of EHEC infection on these physiological functions and the underlying mechanisms will be explored by three Specific Aims. The first Specific Aim is to characterize the effect of EHEC on intestinal epithelial tight junction barrier function. The mechanisms by which EHEC perturbs the TJ barrier will be addressed focusing on the role of cytoskeletal contraction and changes in key TJ-associated proteins. Specific Aim 2 is to define the signaling pathways by which EHEC activates the inflammatory response within intestinal epithelial cells. The events that lead to the activation of NF-kappaB, the upregulation of pro- inflammatory cytokines, such as IL-8, and neutrophil transmigration will be explored. The studies outlined in Specific Aims l and 2 will use the established in vitro cell culture model. Specific Aim 3 is to establish and characterize a murine model of intestinal EHEC infection, The effects of EHEC infection on TJ barrier function as well as epithelial-initiated inflammation will be explored.